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Regulation of beta cell glucokinase by S-nitrosylation and association with nitric oxide synthase.

Rizzo MA, Piston DW - J. Cell Biol. (2003)

Bottom Line: Using quantitative imaging of multicolor fluorescent proteins fused to GK, we found that the dynamic association of GK with secretory granules is modulated through nitric oxide (NO).Our results in cultured beta cells show that insulin stimulates NO production and leads to S-nitrosylation of GK.GK was also found to interact stably with neuronal NOS as detected by coimmunoprecipitation and fluorescence resonance energy transfer.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, 735 Light Hall, Nashville, TN 37232, USA.

ABSTRACT
Glucokinase (GK) activity plays a key role in glucose-stimulated insulin secretion from pancreatic beta cells. Insulin regulates GK activity by modulating its association with secretory granules, although little is known about the mechanisms involved in regulating this association. Using quantitative imaging of multicolor fluorescent proteins fused to GK, we found that the dynamic association of GK with secretory granules is modulated through nitric oxide (NO). Our results in cultured beta cells show that insulin stimulates NO production and leads to S-nitrosylation of GK. Furthermore, inhibition of NO synthase (NOS) activity blocks insulin-stimulated changes in both GK association with secretory granules and GK conformation. Mutation of cysteine 371 to serine blocks S-nitrosylation of GK and causes GK to remain tightly bound to secretory granules. GK was also found to interact stably with neuronal NOS as detected by coimmunoprecipitation and fluorescence resonance energy transfer. Finally, attachment of a nuclear localization signal sequence to NOS drives GK to the nucleus in addition to its normal cytoplasmic and granule targeting. Together, these data suggest that the regulation of GK localization and activity in pancreatic beta cells is directly related to NO production and that the association of GK with secretory granules occurs through its interaction with NOS.

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Association of GK with nNOS can direct GK localization. (A) Endogenous GK, GK-YFP, and C371S GK-YFP were immunoprecipitated from cell lysates using anti-GK antibodies or anti-GFP antibodies for YFP-tagged proteins in combination with agarose-conjugated secondary antibodies. Precipitates were then heated to 37°C for 10 min in the presence of 1 mM DEANO in PBS (lanes 3 and 4) or an equivalent volume of vehicle (DMSO) in PBS alone (lanes 1 and 2). Pellets (lanes 1 and 3) and supernatants (lanes 2 and 4) were analyzed by SDS-PAGE and Western blot using anti-nNOS antibodies. (B) Cells expressing nNOS-CFP and GK-YFP or GK(C371S)-YFP were examined by two-photon microscopy before and after photobleaching with a 514 nm Ar laser (indicated by the hatched region). Average relative fluorescence (n = 6) for cellular CFP (▪) and YFP (□) intensities were plotted versus time. A dotted line was drawn as a reference to indicate prebleach intensity. (C) FRET between nNOS-CFP and either GK-YFP (•) or GK(C371S)-YFP(○) was examined in living cells by two-photon microscopy. FRET ratios were normalized to pretreatment values (100%) before averaging (n = 6) and plotted versus time. Addition of insulin (100 nM) is indicated by the arrow. (D) Cells were cotransfected with GK-YFP and nNOS-CFP-nuc and examined by confocal microscopy. In cells expressing both constructs, GK-YFP is found colocalized with nNOS in the nucleus (red arrows). GK-YFP did not localize to the nuclei of cells that were singly transfected with GK-YFP (white arrows). In the merged panel, the CFP image was colored blue and the YFP image was colored green. Colocalization is indicated by cyan (red arrows).
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fig3: Association of GK with nNOS can direct GK localization. (A) Endogenous GK, GK-YFP, and C371S GK-YFP were immunoprecipitated from cell lysates using anti-GK antibodies or anti-GFP antibodies for YFP-tagged proteins in combination with agarose-conjugated secondary antibodies. Precipitates were then heated to 37°C for 10 min in the presence of 1 mM DEANO in PBS (lanes 3 and 4) or an equivalent volume of vehicle (DMSO) in PBS alone (lanes 1 and 2). Pellets (lanes 1 and 3) and supernatants (lanes 2 and 4) were analyzed by SDS-PAGE and Western blot using anti-nNOS antibodies. (B) Cells expressing nNOS-CFP and GK-YFP or GK(C371S)-YFP were examined by two-photon microscopy before and after photobleaching with a 514 nm Ar laser (indicated by the hatched region). Average relative fluorescence (n = 6) for cellular CFP (▪) and YFP (□) intensities were plotted versus time. A dotted line was drawn as a reference to indicate prebleach intensity. (C) FRET between nNOS-CFP and either GK-YFP (•) or GK(C371S)-YFP(○) was examined in living cells by two-photon microscopy. FRET ratios were normalized to pretreatment values (100%) before averaging (n = 6) and plotted versus time. Addition of insulin (100 nM) is indicated by the arrow. (D) Cells were cotransfected with GK-YFP and nNOS-CFP-nuc and examined by confocal microscopy. In cells expressing both constructs, GK-YFP is found colocalized with nNOS in the nucleus (red arrows). GK-YFP did not localize to the nuclei of cells that were singly transfected with GK-YFP (white arrows). In the merged panel, the CFP image was colored blue and the YFP image was colored green. Colocalization is indicated by cyan (red arrows).

Mentions: Since other proteins that react with NO are known to form complexes containing nNOS (Brenman et al., 1996; Fang et al., 2000; Nedvetsky et al., 2002), we examined the role of nNOS in determining GK localization. To detect interaction between GK and nNOS, we immunoprecipitated endogenous GK from cell lysates and probed for endogenous nNOS by Western blot (Fig. 3 A). We were able to detect nNOS in these precipitates and also from those of expressed GK-YFP and GK(C371S)-YFP. Incubation of GK precipitates with diethylamine nitric oxide (DEANO), a chemical that rapidly releases NO, resulted in elution of nNOS from endogenous GK and GK-YFP precipitates but not from GK(C371S)-YFP precipitates. These data show that GK association with nNOS is both consistent with GK association with secretory granules and also sensitive to the presence of NO.


Regulation of beta cell glucokinase by S-nitrosylation and association with nitric oxide synthase.

Rizzo MA, Piston DW - J. Cell Biol. (2003)

Association of GK with nNOS can direct GK localization. (A) Endogenous GK, GK-YFP, and C371S GK-YFP were immunoprecipitated from cell lysates using anti-GK antibodies or anti-GFP antibodies for YFP-tagged proteins in combination with agarose-conjugated secondary antibodies. Precipitates were then heated to 37°C for 10 min in the presence of 1 mM DEANO in PBS (lanes 3 and 4) or an equivalent volume of vehicle (DMSO) in PBS alone (lanes 1 and 2). Pellets (lanes 1 and 3) and supernatants (lanes 2 and 4) were analyzed by SDS-PAGE and Western blot using anti-nNOS antibodies. (B) Cells expressing nNOS-CFP and GK-YFP or GK(C371S)-YFP were examined by two-photon microscopy before and after photobleaching with a 514 nm Ar laser (indicated by the hatched region). Average relative fluorescence (n = 6) for cellular CFP (▪) and YFP (□) intensities were plotted versus time. A dotted line was drawn as a reference to indicate prebleach intensity. (C) FRET between nNOS-CFP and either GK-YFP (•) or GK(C371S)-YFP(○) was examined in living cells by two-photon microscopy. FRET ratios were normalized to pretreatment values (100%) before averaging (n = 6) and plotted versus time. Addition of insulin (100 nM) is indicated by the arrow. (D) Cells were cotransfected with GK-YFP and nNOS-CFP-nuc and examined by confocal microscopy. In cells expressing both constructs, GK-YFP is found colocalized with nNOS in the nucleus (red arrows). GK-YFP did not localize to the nuclei of cells that were singly transfected with GK-YFP (white arrows). In the merged panel, the CFP image was colored blue and the YFP image was colored green. Colocalization is indicated by cyan (red arrows).
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Related In: Results  -  Collection

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fig3: Association of GK with nNOS can direct GK localization. (A) Endogenous GK, GK-YFP, and C371S GK-YFP were immunoprecipitated from cell lysates using anti-GK antibodies or anti-GFP antibodies for YFP-tagged proteins in combination with agarose-conjugated secondary antibodies. Precipitates were then heated to 37°C for 10 min in the presence of 1 mM DEANO in PBS (lanes 3 and 4) or an equivalent volume of vehicle (DMSO) in PBS alone (lanes 1 and 2). Pellets (lanes 1 and 3) and supernatants (lanes 2 and 4) were analyzed by SDS-PAGE and Western blot using anti-nNOS antibodies. (B) Cells expressing nNOS-CFP and GK-YFP or GK(C371S)-YFP were examined by two-photon microscopy before and after photobleaching with a 514 nm Ar laser (indicated by the hatched region). Average relative fluorescence (n = 6) for cellular CFP (▪) and YFP (□) intensities were plotted versus time. A dotted line was drawn as a reference to indicate prebleach intensity. (C) FRET between nNOS-CFP and either GK-YFP (•) or GK(C371S)-YFP(○) was examined in living cells by two-photon microscopy. FRET ratios were normalized to pretreatment values (100%) before averaging (n = 6) and plotted versus time. Addition of insulin (100 nM) is indicated by the arrow. (D) Cells were cotransfected with GK-YFP and nNOS-CFP-nuc and examined by confocal microscopy. In cells expressing both constructs, GK-YFP is found colocalized with nNOS in the nucleus (red arrows). GK-YFP did not localize to the nuclei of cells that were singly transfected with GK-YFP (white arrows). In the merged panel, the CFP image was colored blue and the YFP image was colored green. Colocalization is indicated by cyan (red arrows).
Mentions: Since other proteins that react with NO are known to form complexes containing nNOS (Brenman et al., 1996; Fang et al., 2000; Nedvetsky et al., 2002), we examined the role of nNOS in determining GK localization. To detect interaction between GK and nNOS, we immunoprecipitated endogenous GK from cell lysates and probed for endogenous nNOS by Western blot (Fig. 3 A). We were able to detect nNOS in these precipitates and also from those of expressed GK-YFP and GK(C371S)-YFP. Incubation of GK precipitates with diethylamine nitric oxide (DEANO), a chemical that rapidly releases NO, resulted in elution of nNOS from endogenous GK and GK-YFP precipitates but not from GK(C371S)-YFP precipitates. These data show that GK association with nNOS is both consistent with GK association with secretory granules and also sensitive to the presence of NO.

Bottom Line: Using quantitative imaging of multicolor fluorescent proteins fused to GK, we found that the dynamic association of GK with secretory granules is modulated through nitric oxide (NO).Our results in cultured beta cells show that insulin stimulates NO production and leads to S-nitrosylation of GK.GK was also found to interact stably with neuronal NOS as detected by coimmunoprecipitation and fluorescence resonance energy transfer.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, 735 Light Hall, Nashville, TN 37232, USA.

ABSTRACT
Glucokinase (GK) activity plays a key role in glucose-stimulated insulin secretion from pancreatic beta cells. Insulin regulates GK activity by modulating its association with secretory granules, although little is known about the mechanisms involved in regulating this association. Using quantitative imaging of multicolor fluorescent proteins fused to GK, we found that the dynamic association of GK with secretory granules is modulated through nitric oxide (NO). Our results in cultured beta cells show that insulin stimulates NO production and leads to S-nitrosylation of GK. Furthermore, inhibition of NO synthase (NOS) activity blocks insulin-stimulated changes in both GK association with secretory granules and GK conformation. Mutation of cysteine 371 to serine blocks S-nitrosylation of GK and causes GK to remain tightly bound to secretory granules. GK was also found to interact stably with neuronal NOS as detected by coimmunoprecipitation and fluorescence resonance energy transfer. Finally, attachment of a nuclear localization signal sequence to NOS drives GK to the nucleus in addition to its normal cytoplasmic and granule targeting. Together, these data suggest that the regulation of GK localization and activity in pancreatic beta cells is directly related to NO production and that the association of GK with secretory granules occurs through its interaction with NOS.

Show MeSH